In recent years, unsaturated fatty acids are noticed not only as essential fatty acids in supplements, but since particularly eicosapentaenoic acid ethyl ester was approved as a therapeutic drug of arteriosclerosis obliterans and hyperlipidemia as switch OTC, there is an expanding market for pharmaceutical-grade EPA ethyl. In other words, highly unsaturated fatty acids have been utilized in pharmaceutical products and health foods. However, since highly unsaturated fatty acids have many double bonds, it is difficult to obtain them with chemical synthesis.
Highly unsaturated fatty acids are produced by extraction and purification from marine organisms such as fish oils. However, contents of highly unsaturated fatty acids are small, and thus a purification technique with a high yield/high purity has been desired.
Conventionally, with regard to methods of separating a specific fatty acid from a mixture of fatty acids or monoesters, there were mainly distillation, molecular sieve, and supercritical fluid extraction as separation methods using the difference in carbon numbers; and low-temperature fractionation, urea addition, silver complex formation, solvent fractionation, and column chromatographic methods using silver ion treatment resin and ODS, as separation methods using the number of double bonds. Although these purification methods are simple, each of them have shortcomings such as insufficiency in separation, and unsuitableness for industrial mass treatment even if high purification is possible, as in the case of chromatographic methods. In actual industrial production, highly pure fatty acids are produced by combining a plurality of these purification methods. However, there are many points to be improved in the complication of the purification methods and in the purity/yield.
The principle of silver nitrate treatment lies on the point that, among highly unsaturated fatty acids, particularly highly unsaturated fatty acids having a large number of double bonds within the molecule form a complex of the double bonds within the molecule and silver ions, by mixture with a silver nitrate aqueous solution. By utilizing this property, analysis on unsaturated fatty acids that utilizes the difference of holding force from a silica gel column carrier carrying silver ions, has been practiced. In the industrial production method of highly unsaturated fatty acids, water solubility of this complex is utilized to fractionate fat-soluble fractions, which include saturated fatty acids or moderately/highly unsaturated fatty acids having a small number of double bonds within the molecule, as oil layers. Since a water-soluble complex can be separated into silver nitrate and purified highly unsaturated fatty acids by heating the solution, such simple process enables purification of highly unsaturated fatty acids in an industrial scale.
However, silver nitrate treatment has drawbacks as a production method in that the price of silver nitrate, which is the raw material, is high, and there are big fluctuations in the price. In addition, since not all of the targeted highly unsaturated fatty acids form a complex, and some of them will remain in the oil layer, it is difficult to say that this is a production method achieving a high yield. As a result, there is a drawback that the production cost is raised.
Furthermore, in principle, there is a difficulty in completely removing highly unsaturated fatty acids having three or more double bonds within the molecule as impurities, in silver nitrate treatment. At the time of high-purity purification achieving pharmaceutical grades, such impurities become the cause of reducing the yield of purification methods in the preceding purification steps and the subsequent purification steps.
Representative purification methods in conventional techniques will be exemplified below.
Problems of techniques that have been used in purification of unsaturated fatty acids and derivatives thereof will be described below.                1) Precision Distillation            Characteristic: a method of separation by utilizing differences in boiling points of each component.    Problem: thermal denaturation may occur; a long time is required in purification of highly pure products.            2) Molecular Distillation            Characteristic: little thermal influence at the time of distillation.    Problem: low separation ability.            3) Urea Treatment Method            Characteristic: utilizing the property of dissolved urea, i.e., incorporating in coexisting linear molecules while forming hexagonal columnar adduct crystals at the time of crystallization.    Problem: low selectivity; waste disposal due to derivation of urea adduct.            4) Silver Nitrate Treatment            Characteristic: utilizing the property of a silver nitrate aqueous solution, i.e., forming a complex with double bonds of fatty acids.    Problem: the price of silver is unstable; involve impurities.            5) Fixed-Bed Chromatography            Characteristic: small thermal influence; separation with high precision is enabled.    Problem: large usage of eluents; unsuitable for industrial production.            6) Simulated Moving-Bed Chromatography            Characteristic: suitable for industrialization due to small usage of eluents and continuous operations.    Problem: ODS fillers, which are often used for separation of unsaturated fatty acids, are expensive; since only single solvent can be used, gradient separation is not possible.
With regard to purification techniques of eicosapentaenoic acid (EPA) ethyl esters, for example, Patent Literatures 1-4 describe silver nitrate treatment as an industrially remarkable purification technique of unsaturated fatty acids and derivatives thereof. However, the methods of Patents 1-4 have problems as follows:                1) unsaturated fatty acids other than EPA and docosahexaenoic acid (DHA) also form a complex at the same time, and it is difficult to obtain highly unsaturated fatty acids with high purity; and        2) some of highly unsaturated fatty acids do not form a complex, and the yield is reduced by disposing them.        
A production method of highly unsaturated fatty acids with a high purity/high yield that compensates for the shortcomings of the conventional techniques is required.